Tumor-associated neutrophils display a distinct N1 profile following TGFβ modulation: A transcriptomics analysis of pro- vs. antitumor TANs

It is becoming increasingly clear that tumor-associated neutrophils (TANs) play an important role in cancer biology, through direct impact on tumor growth and by recruitment of other cells types into the tumor. The function of neutrophils in cancer has been the subject of seemingly contradicting reports, pointing toward a dual role played by TANs in tumor progression. The existence of multiple neutrophil subsets, as well as phenotypic modulation of the neutrophils by various factors in the tumor microenvironment, has been shown. TGFβ plays a significant role in the determination of neutrophils' phenotype, by shifting the balance from an antitumor (N1) toward a more permissive (N2) phenotype. The full range of mechanisms responsible for the pro- vs. antitumor effects of TANs has not yet been elucidated. Therefore, the ability to identify the different neutrophil subpopulations in the tumor is critical in order to understand TANs evolution and contribution throughout tumor progression. Using a transcriptomic approach, we identified alternations in gene expression profile following TGFβ inhibition. We show that N1 and N2 TANs represent distinct subpopulations with different transcriptional signatures and both differ from naive bone marrow neutrophils. The analysis highlights a clear difference in pathways involved in neutrophil function such as cytoskeletal organization and antigen presentation, as well as alterations in chemokine profile, eventually affecting their effect on tumor cells and tumor growth. These data highlights several potential new pathways and mechanisms by which neutrophils can influence both the tumor cells and the adaptive immune system. Tumor immunosuppression is modulated via secretion of inhibitory cytokines as well as the recruitment of specific cell types to the tumor microenvironment. The phenotype of the myeloid cells (neutrophils and macrophages), which represent a key part of this immunosuppressive network, may be altered by the tumor microenvironment to support tumor growth. Despite growing evidence for the presence of multiple neutrophils subpopulations in cancer, the role of neutrophils in tumor growth and metastatic progression has not yet been fully elucidated. Whereas several studies have provided evidence for neutrophils with pro-tumor functions such as promoting tumor angiogenesis, tumor cell dissemination, and metastatic seeding in distant organs, others have shown the existence of neutrophils with antitumor and anti-metastatic functions. These seemingly conflicting reports over the function of neutrophils in cancer is likely due to the existence of multiple neutrophil subsets; a tumor promoting or N2 phenotype and an tumor-inhibitory or N1 phenotype. Interestingly, it has been proposed that there can be phenotypic or functional plasticity, where neutrophils infiltrating a tumor are modulated by cues present in the tumor environment. This has been well exemplified by recent works showing that TANs can be modulated and polarized toward an N1 phenotype by type-1 interferons or a pro-tumorigenic (N2) phenotype by the presence of TGFβ as demonstrated by the observation that systemic inhibition of TGFβ using a specific Alk5 kinase inhibitor called SM16 in mice caused a shift toward a pro-inflammatory and antitumor phenotype (N1). Similarly to tumor-associated macrophages (TAMs), N2 TANs can exert their effect by secreting pro-tumor factors and by affecting other cells of the immune system in an immunosuppressive manner, for example, by inducing T-cell tolerance. Accordingly, depletion of N2-polarized TANs inhibits tumor growth and metastasis and reduces the level of immunosuppression in the tumor microenvironment, allowing for increased activity of CD8+ cytotoxic T-lymphocytes (CTL). In contrast, in the presence of type-I IFNs or absence of TGFβ, TANs display an antitumor N1 phenotype with increased tumor cytotoxicity, high neutrophil extracellular traps (NETs) expression, high ICAM1, and TNF-α expression. Using transcriptomic analysis, we have previously shown that N2-polarized TANs display a dramatically different transcriptomic profile than both myeloid-derived suppressor cells (G-MDSCs) and naive bone marrow neutrophils (BMN), including the expression of a wide range of cytokines (CCL17, CCL2, CCL5). By secreting CCL17, TANs were further shown to recruit regulatory T-cells to the tumor site, thus inducing immunologic self-tolerance and impaired immune response to tumor cells. Conversely, inhibition of TGFβ-driven N2-polarization was shown to promote TANs' expression of cytokines such as CCL3 and TNFα, further attracting CD8+ T cells to the tumor site. Since no definitive markers discriminating between pro-tumor (N2) and antitumor (N1) neutrophils have yet been established, the identification of neutrophils subsets in the tumor remains a challenge, limiting our understanding of the evolution TANs and their specific contribution throughout tumor progression. The purpose of this study was to gain a deeper understanding of N1 and N2 TANs' polarization states using a transcriptomics approach and to identify the genes and pathways modified following their polarization, resulting into their opposite anti or pro-tumor effect. We examined which pathways and gene-groups varied among these two populations of neutrophils and performed a detailed analysis of pathways related to the main functions of neutrophils, such as phagocytosis, antigen presentation, and specific immune effects, as well as response to stress and structural genes. Our data reveal a significant difference in chemokine/cytokine signature between N2 and N1 TANs, which might further help understanding the chemotaxis forces and role of neutrophils in tumor. Finally, we examined the differential functional effects of N1 and N2 neutrophils on tumor growth, finding that the overall N1 signature is cytotoxic to tumor cells, whereas N2 neutrophils contribute to tumor growth.